Method and apparatus for precise selection and extraction of a focused component in isoelectric focusing performed in micro-channels

ABSTRACT

An apparatus and method are disclosed for the precise selection and extraction of a selected analyte in a focused zone produced by isoelectric focusing performed in micro-channels. A cross-channel microfluidic device comprises a sample mixture introduction and separation channel and an extraction channel, which are in fluid communication with each other at a point of intersection. Means are provided for selectively moving the pattern of separated zones following cIEF to the intersection point, and means are provided for applying an extraction pressure to direct a single zone containing a selected analyte into and then out of the extraction channel for collection.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.Provisional Patent Application No. 60/819,390, filed on Jul. 10, 2006,the disclosure of which is incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

N/A

FIELD OF THE INVENTION

This invention relates to the separation and precise selection andextraction of components of an analyte mixture by means of liquid-phasecapillary or micro-channel electrophoresis. The principal application ofthe invention is to facilitate the identification and characterizationof the extracted component(s) through the subsequent use ofmicro-analytical techniques such as mass spectrometry.

BACKGROUND OF THE INVENTION

Capillary electrophoresis (CE) has been established as an importantseparation technique in bioanalytical chemistry. Separation anddetection of very small amounts of biological samples, about pL-nLvolumes, can be achieved with CE. This is generally not possible withmore conventional separatory methods, even high-performance liquidchromatography (HPLC). There are several CE separation modes in use fordifferent kinds of samples. They include capillary zone electrophoresis,moving boundary capillary electrophoresis, capillary isotachophoresisand capillary isoelectric focusing (cIEF).

CE provides high-resolution and high efficiency separation and is usedin proteomic research and biopharmaceutical applications. Not onlyproven as a powerful analytical tool, CE is also promising forapplication in nano and micro-fractionation collection. For instance,on-line coupling of CE with mass spectrometry (MS) is used to elucidateprotein structure, and off-line CE fractionation collection is importantfor further characterization of proteins in connection with sequencing,peptide digesting and mapping and reaction studies.

Various designs of microfluidic apparatus have been used for CE fractioncollection. Vial collection and membrane collection of individualcomponents of an analyte at the exit of the separation column, with orwithout the help of sheath fluid, has been investigated. Inpoint-detection capillary electrophoresis, sample fractions arecollected from the outlet of the separation channel after passing thedetection window. Karger et al. [1] further developed vial collection toa fraction collector. As CE is normally run with 25 to 75 μm i.d.capillaries or micro-channels, extremely small volumes of individualfractions can be expected. Therefore, exact timing is important forprecise fractionation. Cross-contamination frequently occurs for closelymigrating peaks due to the extremely narrow peak width and extremelysmall amount of eluant.

An important CE mode, capillary isoelectric focusing (cIEF), is used forseparating amphoteric substances such as peptides and proteins in acapillary or micro-channel under an electric field. Across theseparation capillary or channel, voltage is applied and a pH gradient iscreated by carrier ampholytes that have been pre-mixed with the analytesample, acidic at the anodic end of the channel and alkaline at thecathodic end of the channel. Each component in the analyte mixturemigrates to a position in the separation channel where the surroundingpH corresponds to its isoelectric point. Therein, as zwitterionspossessing no net charge, molecules of that component cease to move inthe electric field. Different amphoteric components are thereby focusedinto narrow, stationary zones.

cIEF is the highest resolution CE mode for charge-based separation ofamphoteric substances such as proteins and peptides. It has most oftenbeen used to separate closely related proteins having subtle differencesbetween their structures. In the cIEF separation channel, components ofthe analyte mixture, which evenly distribute along the whole channelbefore the separation process, are separated and focused into narrow,stationary, component zones.

Uniquely narrow zones are formed using cIEF because: 1) zone broadeningdue to parabolic flow is not a factor in the separation process; 2) thefocusing force is reverse to diffusion and 3) the electrophoresiscurrent during focusing is low compared to other CE modes thusminimizing the effects of component zone broadening due to Jouleheating. For these reasons, the analyte components from cIEF areconcentrated over a hundredfold in their separated, narrow zones.

In column or micro-channel separation technologies, the narrower thecomponent zone is the higher the resolution. Narrow zones in other CEmodes can be achieved by injecting a very small ‘analyte plug’representing a very small segment of the whole separation channel. Eventhen, however, component zone broadening is unavoidable during theseparation process for the reasons stated above. By contrast, cIEFallows the whole separation channel to be filled with the analyte samplemixture without any deterioration of the separation resolution. Incomparison to other CE modes, this provides cIEF with a much higheranalyte loading capacity.

The absence of parabolic flow broadening with static, focused zones andlow electropheresis current makes cIEF's separation resolution much lessdependent on small dimensional separation channels. The separationchannel's cross-sectional dimension can be 2 to 5 times larger thanother CE modes with comparable separation resolution. Use of a largercross-sectional separation channel again further increases sampleanalyte loading capacity. Higher sample loading provides a higher amountof extracted component material. Increasing the amount of extractedmaterial is highly desirable since it generally increases the analyticalsuccess of subsequent analytical techniques such as mass spectrometry.

For the foregoing reasons, cIEF would appear to be an attractivetechnique for nano/micro preparative fractionation of closely relatedproteins from a mixture, for example, variants of hemoglobin arisingfrom mutations to the amino acids sequences; or different forms ofrecombinant proteins arising from the heterogeneity associated withdifferent post-translational modifications.

Others have investigated the use of cIEF for fraction collection, as anaspect of CE fractionation generally. For example, Guttman et al [4,5]have studied “planar” electrophoresis using a capillary cross-connector.By applying different voltage configurations through differentreservoirs, a component zone or peak was collected after passing asingle-point detector.

To date, however, others have had limited success in achieving any highdegree of precision in the selection and extraction of cIEF componentzones, chiefly because of the following limitations to the operation oftheir devices: 1) mobilization flow is in one direction; 2) mobilizationspeed is pre-determined and generally fixed and 3) inability tovisualize the entire separation zones and detect in real-time anyzone-width distortion due to mobilization.

In conventional “single point on-column” detection cIEF, the focusedzones or peaks within the capillary must be moved, chemically orelectroosmotically, past the detection point to be detected. Thismobilization step in cIEF requires extra time and distorts the focusedpeaks, making it difficult to collect pure peaks withoutcross-contamination when peaks focus in close proximity.

It has, accordingly, not been possible with existing microfluidic(capillary) electrophoretic devices for micro-preparative fractioncollection to observe all of the separated peaks developed byelectrophoresis, then select a particular peak of interest and mobilizethe entire pattern of peaks, while maintaining or re-establishing thefocus of the pattern to bring the selected peak to aseparation/collection point.

It is a principal object of the current invention to provide anintegrated micro-scale electrophoresis device (IMED) for the extractionof large (μg) or small (ng) amounts of components (in particular,proteins) separated by cIEF.

It is a particular object of the invention to provide such an IMED asaforesaid which is adapted for automatic sample injection and capable ofhigh-resolution protein separation, selection of a specific protein zoneor peak and precision extraction of the central portion of the selectedprotein peak (“heart-cut extraction”) for further characterization.

SUMMARY OF THE INVENTION

In its broadest aspect, the apparatus of the invention is apparatususeful for the selective extraction of an analyte from a mixture ofanalytes using capillary isoelectric focusing, which includes: acapillary separation channel filled with a medium containing a mixtureof ionic components in which target analytes migrate are separated intozones and in stationary or near stationary equilibrium; a capillaryextraction channel intersecting with and angularly displaced from saidcapillary separation channel and in fluid communication therewith at thelocation of intersection; means for causing selected zones of analytesseparated by capillary isoelectric focusing to move to said intersectionof the separation and extraction channels in a time-independent manner;optical whole column imaging detection apparatus for monitoring theisoelectric focusing process and observing the position of the separatedzones of analyte; and means for applying an extraction force to direct asingle zone containing a selected analyte into and then out ofextraction channel in a time-independent manner.

According to a preferred embodiment of apparatus according to to theinvention there is provided a coplanar, cross-channel microfluidicdevice comprising a sample mixture introduction and separation channeland an extraction channel in fluid communication at a point ofintersection; means of producing a pattern of amphoteric componentsseparated into focused, stationary zones within the separation channel;an apparatus for irradiating the whole separation channel withultraviolet (UV) light and having a detector that can provide UVabsorption imaging detection of the whole separation channel providingreal-time or at least very, rapid digital images of the cIEF separationprocess; means of selecting a specific focused, component zone or peakin the separation channel and moving the said peak to the intersectionof the separation and extraction channels; the aforementionedwhole-channel, real-time imaging detection apparatus to monitor themovement of the selected component peak and to visualize the alignmentof said peak to the intersection of the separation and extractionchannels; and means for moving the aligned, component peak or a portionof the peak into and then out of the extraction channel for collectionor interface to a second analytical apparatus such as a massspectrometer.

The invention is also directed to a method for fractionating andextracting analytes capable of resolution by capillary isoelectricfocusing, comprising the steps of providing a capillary separationchannel and a capillary extraction channel in fluid communicationtherewith; introducing an analyte sample containing a mixture of ionicanalytes prepared for electrophoresis into the separation channel;separating and focusing components of the analyte mixture into separatedzones in the separation channel using capillary isoelectric focusing;monitoring the process of capillary isoelectric focusing and positionsof the separated analyte zones using whole column imaging detectionmeans; causing the separated zones to move selectively to theintersection of the separation and extraction channels; and applying anextraction force to direct a zone containing desired analytes outthrough the extraction channel for collection.

According to particular embodiments of the method of the invention, theseparated zones are caused to move along the separation channel to theintersection of the separation and extraction channels by orienting theseparation channels so that gravity causes the desired motion.Microfluidic delivery means may be used to move the separated zones andalso to apply the extraction force.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described further, with reference to theaccompanying drawings, in which:

FIG. 1 is a schematic illustration of essential components of an IMEDfor use in the separation and extraction of sample components accordingto the present invention;

FIG. 2 schematically illustrates an IMED of the same configuration asFIG. 1, but illustrating the arrangement of electrolyte holding tanksand semipermeable membranes used for handling of ampholytes and analytesamples in solution into the IMED;

FIG. 3 illustrates a variant of the IMED of FIG. 2, having a differentconfiguration of interconnecting separation and extraction channelcapillaries;

FIG. 4 is a graphical representation (electropherograms) of proteinzones as light absorbance peaks along the whole length of the separationchannel, illustrating the separation, manipulation and extraction ofselected component Hemoglobin C by means of the IMED of FIG. 3;

FIG. 5 presents electropheregrams of absorbance peaks versus separationchannel position showing the separation and extraction of selectedcomponent Hemoglobin S in the same IMED as employed to produce theresults of FIG. 4;

FIG. 6 shows the cIEF electrophoregram of myoglobin and transferrineusing an IMED having the structure shown in FIG. 2;

FIG. 7 illustrates the mass spectrogram of separated and extractedmyoglobin component from a separation carried out by apparatus of theinvention and illustrated in FIG. 3; and

FIG. 8 illustrates the mass spectrogram of separated and extractedtransferrine component from the separation carried out by apparatus ofFIG. 3.

DESCRIPTION OF PREFERRED EMBODIMENT

As illustrated in FIG. 1, an analyte sample containing a mixture ofionic components is prepared for capillary isoelectric focusing (cIEF)electrophoresis and is injected into a separation channel 10 (horizontalin the Figure) in fluid communication with an extraction channel 12(vertical in the Figure) until it is completely full (overflowing). Inthe separation channel, components of the sample mixture are separatedand focused using cIEF. Channels 10 and 12 may be in the form of aco-planar, monolithic microfluidic device or microchip in which closed,elongate separation and extraction channels have been created. In analternative arrangement of channels 10 and 12, their locus ofintersection and fluid communication may be formed by four pieces offused silica capillary tubing.

A high voltage power supply 14 is connected to anode 16 and cathode 18and sets up both a voltage and a pH gradient liquid medium, acidic inthe region of anode 16 and alkaline near cathode 18. As noted above,each component in the analyte mixture migrates to a positioncorresponding to it isoelectric point. Different amphoteric compoundsare thereby focused into narrow, stationary zones.

FIG. 2 is operationally equivalent to the IMED of FIG. 1, but furthershows two electrolyte tanks 22 a and 22 b that are integrated in theIMED to hold anolyte and catholyte. These tanks are connected with theseparation channel through semi-permeable membranes 23 a and 23 b. Thesemi-permeable membrane, having a thickness of about 10 μm and a poresize less than 10 nm is sandwiched between the separation channel andthe electrolyte tank, to permit the application of high level electricfields and to avoid unwanted mixing of the sample with electrolytes inthe electrolyte tanks.

In the arrangement of FIG. 2, separation and extraction channels 10 and12 are formed in a microchip 24 and respectively extend beyondperpendicular edges of the microchip 24 through capillaries 10 a/10 band 12 a/12 b.

When the IMED illustrated in FIG. 2 is used to extract proteins, theentire separation channel is UV transparent to allow whole separationchannel UV detection.

According to the invention, the surface properties (e.g., hydrophobicityand surface charge) of at least the separation channel of the IMED areselected with a view to achieving high resolution separation ofamphoteric components such as proteins.

An important reason for controlling the surface charge in the separationchannel is to minimize electro-osmotic flow. Many plastic substratessuch as polycarbonate, Teflon® and PMMA exhibit sufficient transparencyto ultraviolet light for the purposes of the invention and developlimited surface charge. With dynamic coating using methyl cellulose(MC), MC derivatives or polyvinyl alcohol, the separation channelsurface is unreactive with protein.

If UV transparent fused silica, glass or quartz is employed as theseparation channel substrate for the IMED, additional surfacemodification is necessary to control surface charge. This can beachieved by coating with such polymers as linear or cross-linkedpolyacrylamide.

As seen in FIG. 2, separation channel 10 is connected with two pieces ofcapillary 10 a and 10 b at either end, to allow for the injection ofsample and for manipulation of the separated component peaks. Separationchannel 10 crosses integrally with extraction channel 12, whosedimension should be no larger than that of the separation channel toallow for “heart cut” extraction of the selected component peak. As withthe separation channel 10, extraction channel 12 has two integralcapillaries 12 a and 12 b communicating with either end of extractionchannel 20, to allow for peak extraction and collection or interfacewith a second selected separation/analytical apparatus, such as a massspectrometer.

FIG. 3 shows an arrangement like that of FIG. 2, but in whichcapillaries 12 a and 12 b extend perpendicularly to their associatedextraction channel 12 as does extension capillary 10 a relative toseparation channel 10.

The apparatus of the invention includes a whole column imaging device(WCID) apparatus [not shown in the drawings] to irradiate the whole orat least a large portion of the length of the separation channel withultraviolet (UV) light or other light. Apparatus of this kind has beendescribed in United States patents of some of the present inventors,particularly U.S. Pat. Nos. 5,784,154 and 6,852,206, whose disclosuresare hereby incorporated by reference. The WCID incorporates a detectorthat provides UV absorption or fluorescent imaging detection of thewhole, or at least a large portion, of the length of separation channel.The apparatus is capable of providing real-time or at least very, rapiddigital absorbance images of the separation channel and incorporatesimaging optics (e.g., lens, CCD) that afford imaging resolutions thatexceed the separation resolution of the cIEF method.

During the entire cIEF separation and focusing process, the whole (ornear whole) separation channel is monitored using the aforementionedreal-time image detection apparatus and the complete componentseparation pattern within the separation channel is ‘visualized’. At anygiven moment, the precise spatial position and width of each componentzone or peak in the separation channel is known.

After the cIEF separation and focusing process is complete, theseparated zones or peaks are stationary and remain spatially fixedrelative to each other. These focused peaks, as a set, can be moved backand forth (i.e., to the right or to the left within the horizontalseparation channel) with extremely fine axial resolution control. Forexample, axial motion control of a peak can be less than +/−50 um withnegligible peak-broadening distortions using a number of ways such aspressure difference. Axial motion control of this accuracy requiresreal-time (or near-real time) monitoring using the above detectionapparatus that visualizes the whole length of the separation channel.Also, the exact spatial position and width of the extraction channelthat intersect the separation channel can be determined by thewhole-channel detection apparatus.

With particular reference to FIG. 1, separation channel 10 andextraction channel 12, are in fluid communication where they intersect.Before sample mixture introduction, the extraction channel 12 iscompletely filled (overflowing) with a desired matrix (such as water ora selected pH and ionic strength buffer), and then both ends are sealedor plugged (e.g., with end caps or shut valves). This sealing constrainsthe matrix within the sample extraction channel and also prevents themixing of the sample mixture during sample mixture introduction. Theseparation channel 10 is then completely filled (overflowing) with theanalyte sample containing a mixture of ionic components is prepared forcIEF separation. Upon application of an electric field across theseparation channel, analyte focusing and separation is conducted withinthe separation channel, which is visualized or monitored with areal-time, whole-separation channel detection apparatus.

If identification of a particular analyte zone, say Zone A in FIG. 1, isthe objective, then all the zones within the separation channel aremoved slowly toward the left end of the channel by operation of apressure differential imposed in the direction X. During this motion,the electrophoresis voltage is kept on to maintain the focused zones.Once again, the real-time whole-channel detection apparatus is used tovisualize the motion of the zones or peaks towards the left end of thechannel.

The zone or peak pattern can be moved back and forth along theseparation channel with very fine motion control by positive or negativepressure. For example, if one end of the horizontal separation capillaryis slightly raised or lowered with respect to the other end, the peakpattern will move under the force of gravity. Alternatively, a pressuredifferential can be created across the separation channel by means of aconventional microfluidic delivering system, in the nature of a pump.The invention also contemplates movement of the peak pattern back andforth by chemical means, e.g. by changing the composition of the anolyteor the catholyte (located in tanks at each end of the separationchannel).

Once Zone A is aligned with the extraction channel 12 intersection(e.g., the centerline of Zone A is superimposed over the centerline ofthe extraction channel) as can be visualized by the real-time, detectionapparatus, the imposed lateral movement of Zone A is stopped. Thisextraction method is time independent and adaptive in nature in that itallows for very fine corrective or adjustment motion in either directionuntil the selected zone is properly aligned to the extraction channel.

With the selected zone now properly aligned at the intersection of theseparation and extraction channels, both ends of the separation channel10 are sealed or plugged (e.g., with end caps or shutoff valves) andboth ends of the extraction channel 12 are unplugged or unsealed. Zone Aextraction is performed by applying a controlled pressure differenceacross the ends of the extraction channel until Zone A is passed intoand then out of the extraction channel. Again, the pressure differentialmay simply be the force of gravity or be established by the pressureimpressed across the channel by micro-fluidic delivering system; or, bysetting up an electro-osmotic flow—EOF (applying voltage through twomicrovials at both ends of the extraction channel, with the channel wallmodified to generate EOF in the desired direction). In this manner, overshort distances, any zone within the separation channel can be selectedand precisely extracted.

During zone or peak motion, the real-time, whole-column detectionapparatus can monitor and visualize for possible separation resolutiondisturbances allowing for corrective actions such as halting motion fora period of time to allow for refocusing.

As noted above, others have used cross-channel arrangements for CE. Thedifferences between the apparatus and method of the present inventionfrom previous attempts at separation and fractionation of analytes,which make our invention more precise for the selection and extractionof an analyte, will now be discussed.

The current invention uses a real-time, whole separation channeldetector apparatus to visualize the entire separation channel. Since theposition of the extraction channel intersection and the position andwidth of the selected component zone are known or are visualizedprecisely at any given time and over short distances very fine axialmotion control can be made using microgravity differences, the alignmentand extraction of the selected zone can be made with high precision andspecificity.

The component zones or peaks within the separation channel can bemicro-manipulated to move back and forth in the separation channel incIEF in an essentially time-independent manner, and this permitscontinuous corrective action until alignment is complete. Combining thestatic nature of cIEF focused peaks with real-time, whole-columndetection makes this method highly selective since any peak in theseparation channel can be selected and moved to the position of theextraction channel. In other methods, all sample peaks move in onedirection and once a peak is missed, it cannot be moved back.

The method and apparatus of the present invention lends itself toautomated peak extraction. The real-time, whole-column detectionapparatus displays absorption peaks on a monitor for humaninterpretation and manipulation for specific peak extraction. However,the electronic signals produced by the real-time, detection apparatuscan be interpreted by a peak-find processing software algorithms andthen, using real-time data control the selection, alignment andextraction of peak based on user-defined extraction criteria.

This method and apparatus are also amenable to multiple cross-channelseparations and extraction channels in a single microchip to assist inautomation.

This method and apparatus could be used to integrate multiple extractionchannels with a single separation channel. Also, the position of anextraction channel intersection can be preferentially located at a placeon the separation channel to optimize extraction of components withcertain pI values.

This method and apparatus of the invention produce extraction volumesand concentrations levels well exceeding the minimum requirement forfurther peak identity analysis of the peak using modern massspectrometry with ionization techniques such as electrospray and matrixassisted laser desorption ionization (MALDI).

EXPERIMENTAL EXAMPLES Example 1

An IMED having the structure shown in FIG. 3 was used to selectivelyextract different hemoglobin variants of a mixture. An imaged cIEFsystem (Model iCE280, Convergent Bioscience Ltd., Toronto, Canada) wasused as the real-time, whole separation channel UV detection apparatus,and protein detection was achieved through UV absorption at 280 nm. Anautosampler from Prince CE system (Prince. Technologies, Emmen, TheNetherlands) was used for sample injection and fluid manipulation alongthe separation channel. Methyl cellulose (MC), hourse heart myoblobin,human transferrin, and pH 3-10 Pharmalyte were obtained from Sigma (StLouse, Mo.). Hemoglobin control AFSC was obtained from HelenaLaboratories (Beaumont, Tex.). HPLC grade water was from J. T. Baker(Phillipsburg, N.J.), and was used for all solutions. Sample mixturesolution was prepared by mixing protein with 8% pH 3-10 Pharmalytes and0.35% MC.

The extraction channel of the IMED was first filled with HPLC water orpreferred buffer, and each end of the capillary connected with theextraction channel was sealed with an end cap or shutoff valve. Then,sample mixture solution was injected into the separation channel of theIMED with the autosampler. Anolyte of 80 mM H₃PO₄ in 0.1% MC andcatholyte of 100 mM NaOH in 0.1% MC were filled into the correspondingelectrolyte tank. cIEF was achieved by applying 1.5 kV across theelectrolyte reservoirs for about 10 minutes and then maintaining at 3 kVduring focusing, peak manipulation, and peak extraction.

FIG. 4 illustrates the separation, manipulation, and extraction ofHemoglobin C achieved in this operation of the IMED. The separatedprotein peaks were monitored with the whole separation channel detector.Trace 1 shows the target components (Hemoglobin C) is located to theleft of the extraction cross section (which is indicated with thevertical line in the figure). The separated protein peaks are directedto the right by an imposed pressure differential. Trace 2 shows thetarget component is to the right of the extraction cross section. Trace3 illustrates how the target component is manipulated previously to theextraction cross section as desired.

At this stage, both ends of the separation channel were shut off andpressure different was applied to the end of extraction channel toextract Hemoglobin C. Trace 4 shows the refocusing of protein peaks witha heart cut Hemoglobin C extracted. It can be seen that the four majorcomponents of Hemoglobin AFSC control are very well separated, while thepeak area of Hemoglobin C is significantly reduced.

This example above illustrates the power of the IMED of the invention inmanipulating separated protein peaks back and forth along the separationchannel to achieve precise heart cut extraction. However, it will seldombe necessary to manipulate a target component back and forth (i.e. intwo linear directions) to position the peak cross section for a preciseheart cut extraction, since the detector allows real time (images atless than 1 second intervals) monitoring of the manipulation process.

Example 2

FIG. 5 shows the separation and extraction of Hemoglobin S in the IMEDwith the device according to FIG. 4. The pI difference betweenHemoglobin F and S and between Hemoglobin S and C was about 0.1. It canbe seen that the separation pattern was not affected significantly uponhemoglobin s extraction, while the peak area of Hemoglobin S was reducedfollowing extraction.

Example 3

An IMED having the structure substantially represented in FIG. 2 wasused to separate and selectively extract target protein component foroff line MALDI-TOF characterization. FIG. 6 shows the cIEFelectrophoregram of myoglobin and transferrine in 8% 5-8 Pharmalytes,0.35% MC solution. It can be seen that the major component oftransferrine and the major component of myoglobin were very wellseparated. Upon cIEF separation, the extracted individual protein wasdiluted to 100 μL in DI water, and 1 μL of this diluted sample wasfurther diluted at either 1:2 or 1:10 ratio with 0.2% (v/v)trifluoroacetic acid in water. The further diluted protein sample of 1μL was mixed with 1 μL of sinapinic acid (10 mg/mL), and 1 μL of thissample mixture was spotted for MALDI TOF MS (Voyager STR, AppliedBiosystems, Foster City, Calif.).

FIGS. 7 and 8 shows the mass spectrograms of extracted myoglobin andtransferrine. From the mass spectrogram, it can be seen that thepresence of carrier ampholytes and MC has insignificant impact on theMALDI TOF MS analysis of protein, and the purity of the extractedprotein was further confirmed.

The above experimental examples demonstrate that the extracted componentin a relatively larger dimension IMED is sufficient for further proteinenzyme digestion and mass spectrometer (MS) characterization.

The foregoing description of embodiments of the invention are forillustrative purposes and no limitation of the invention to the specificconstruction shown and described herein should be inferred. Variousmodifications apparent to those skilled in the art may be made withoutdeparting from the invention defined in the appended claims.

REFERENCES

The disclosure of every reference listed below is hereby incorporatedinto the present specification by reference.

-   -   1. Müller O.; Foret S.; and Karger, B. L. Anal. Chem. 1995, 67,        2974-2980    -   2. Paitchett J. Thomas Electrophoresis 1996, 17, 1195-1201    -   3. Kuhn R.; Hoffsterrer-Kuhn S. Capillary Electrophoresis        Principles and Practice, Springer Laboratory, Berlin Heidelberg,        Germany, 1993    -   4. Khandurina J.; Guttman A. Journal of Chromatography A,        979 (2002) 105-113    -   5. Khandurina J.; Chovain T.; Guttman A. Anal. Chem. 2002 74,        1737-1740    -   6. Herr, Amy E; Molho, Joshua I; etc Anal. Chem. 2003, 75,        1180-1187    -   7. Kniansky, Dusan; Masar, Marian; Anal. Chem. 2000, 72,        3596-3604    -   8. Rocklin, Roy; Ramsey, Roswitha S; Ramsey, J. Micheal Anal.        Chem. 2000, 72, 5244-5249

1. Apparatus for selective extraction of an analyte from a mixture ofanalytes using capillary isolelectric focusing comprising: a capillaryseparation channel filled with a medium containing a mixture of ioniccomponents in which target analytes are separated into zones instationary or near stationary equilibrium; a capillary extractionchannel intersecting with and angularly displaced from said capillaryseparation channel and in fluid communication therewith at the locationof intersection; means for causing selected zones of analytes separatedby capillary isoelectric focusing to move to said intersection of theseparation and extraction channels in a time-independent manner; opticalwhole column imaging detection apparatus for monitoring the isoelectricfocusing process and observing the positions of the separated zones ofanalyte; and means for applying an extraction force to direct a singlezone containing a selected analyte into and then out of the extractionchannel in a time-independent manner.
 2. Apparatus according to claim 1,wherein said intersecting separation channel and said extraction channelare coplanar and are formed in a monolithic microfluidic device ormicrochip as elongate separation and extraction channels.
 3. Apparatusaccording to claim 2, wherein said separation channel is made of aUV-transparent plastic.
 4. Apparatus according to claim 3, wherein saidUV transparent plastic is selected from the group consisting ofpolycarbonate, polyfluorinated polyethylene and polyethylmethacrylate.5. Apparatus according to claim 1, wherein said intersecting separationchannel and said extraction channel intersect at right angles and areformed of four pieces of silica, glass or quartz tubing coated withlinear or cross-linked polyacrylamide.
 6. Apparatus according to claim5, wherein said means for causing selected zones or analytes to move tosaid intersection of the separation and extraction channels in atime-independent manner is operable to tilt said separation channel froma horizontal orientation to allow gravitational force to move said zonesalong the channel.
 7. Apparatus according to claim 5, wherein said meansfor applying an extraction force is operable to raise or lower slightlyone end of the extraction channel relative to the other end, therebycausing the zone to move into the extraction channel under themicroforce of gravity.
 8. Apparatus according to claim 1, wherein saidmeans for selectively causing separated zones to move to theintersection of the separation and extraction channels and said meansfor applying an extraction pressure each comprises a microfluidicdelivering system.
 9. Apparatus according to claim 6, wherein said meansfor applying an extraction pressure comprises a microfluidic deliveringsystem.
 10. Apparatus for the precise selection and extraction of ananalyte from a mixture of ionic analytes, separated by capillaryisoelectric focusing comprising: a capillary separation channel having asample inlet end and an outlet end and having at each end an electrolyteholder in fluid communication with the separation channel through asemi-permeable membrane; a capillary extraction channel angularlydisplaced from said capillary separation channel and in fluidcommunication therewith at a location of intersection and having anextraction outlet end and an inlet end; means for filling the capillaryseparation channel with a mixture of ionic analytes and for causingdifferent analytes to separate and focus into stationary or nearstationary zones by capillary isoelectric focusing electrophoresis;means for causing a selected analyte zone of the analyte zones separatedby capillary isoelectric focusing to move to said intersection of theseparation and extraction channels in a time-independent manner; anoptical image detection apparatus including a light source forirradiation of the whole or at least a large portion of the separationchannel with ultraviolet (UV) light or other light and capable ofacquiring real-time, or at least very rapid, digital images ofultraviolet absorption or fluorescence detection of focused analytezones simultaneously in the whole, or at least a large portion, of theseparation channel; and means for applying an extraction force to directthe selected analyte zone or a portion thereof into and then out of theextraction channel for collection or interface to a second analyticalapparatus.
 11. A method for fractionating and extracting analytescapable of resolution by capillary isoelectric focusing comprising thesteps of: providing a capillary separation channel and a capillaryextraction channel in fluid communication therewith; introducing ananalyte sample containing a mixture of ionic analytes prepared forelectrophoresis into the separation channel; separating and focusingcomponents of the analyte mixture into discrete zones in the separationchannel using capillary isoelectric focusing; monitoring the process ofcapillary isoelectric focusing and the positions of the separatedanalyte zones using whole column imaging detection; causing theseparated zones to move selectively to the intersection of theseparation and extraction channels; and applying an extraction force todirect a zone containing desired analytes into and then out through theextraction channel.
 12. A method according to claim 11, wherein theseparated zones are caused to move selectively to the intersection ofthe separation and extraction channels by orientation of the separationchannel so that gravity causes the analyte sample to move along theseparation channel.
 13. A method according to claim 11, whereinmicrofluidic delivery is used to move the separated zones to theintersection of the separation and extraction channels.
 14. A methodaccording to claim 11, wherein said extraction zone is applied using amicrofluidic delivery pump.
 15. A method according to claim 11, whereinsaid analytes comprise a mixture of proteins.
 16. Method for preciseselection and extraction of an analyte from a mixture of ionic analytesseparated by capillary isoelectric focusing comprising the steps of:providing a capillary separation channel and a capillary extractionchannel in fluid communication therewith; filling to overflow theextraction channel with a matrix buffer such as water or a selected pHand ionic strength buffer and then sealing the ends of the extractionchannel; filling to overflow the separation channel with a samplecontaining a mixture of ionic analytes prepared for isoelectric focusingelectrophoresis; separating and focusing components of the analytemixture into discrete zones in the separation channel using capillaryisoelectric focusing; monitoring in real-time the process of capillaryisoelectric focusing and positions of the separated analyte zones untilthe focusing is complete and focused zones are stationary or nearstationary, using whole separation channel imaging detection; usingreal-time, whole separation channel imaging detection, selecting ananalyte zone, causing said selected analyte zone to move and align withthe intersection of the separation and extraction channels and detectingsaid alignment; sealing the ends of the separation channel; andunsealing the ends of the extraction channel and applying an extractionforce to direct the selected analyte zone or a portion thereof into andthen out of the extraction channel for collection or interface to asecond analytical apparatus.